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Keywords
Autoimmune hemolytic anemia Autoantibodies
Cytokines
Summary
Autoimmune hemolytic anemia (AIHA) is caused by the
increased destruction of red blood cells (RBCs) by antiRBC autoantibodies with or without complement activation. RBC destruction may occur both by a direct lysis
through the sequential activation of the final components of the complement cascade (membrane attack
complex), or by antibody-dependent cell-mediated cytotoxicity (ADCC). The pathogenic role of autoantibodies
depends on their class (the most frequent are IgG and
IgM), subclass, thermal amplitude (warm and cold
forms),as well as affinity and efficiency in activating
complement. Several cytokines and cytotoxic mechanisms (CD8+ T and natural killer cells) are further involved in RBC destruction. Moreover, activated macrophages carrying Fc receptors may recognize and
phagocyte erythrocytes opsonized by autoantibodies
and complement. Direct complement-mediated lysis
takes place mainly in the circulations and liver, whereas
ADCC, cytotoxicity, and phagocytosis occur preferentially in the spleen and lymphoid organs. The degree of
intravascular hemolysis is 10-fold greater than extravascular one. Finally, the efficacy of the erythroblastic compensatory response can greatly influence the clinical picture of AIHA. The interplay and relative burden of all
these pathogenic mechanisms give reason for the great
clinical heterogeneity of AIHAs, from fully compensated
to rapidly evolving fatal cases.
2015 S. Karger GmbH, Freiburg
Introduction
Autoimmune hemolytic anemia (AIHA) is defined as the increased destruction of red blood cells (RBCs) in the presence of
anti-RBC autoantibodies with or without complement activation.
Autoantibodies are produced by both tissue and circulating selfreactive B lymphocytes, following cooperation with T helper lymphocytes. A third key player in anti-RBC autoimmunity is the
complement system, which is able to induce a direct erythrocyte
osmotic lysis through the sequential activation of the membrane
attack complex (MAC). Moreover, autoantibodies are able to destroy RBCs by antibody-dependent cell-mediated cytotoxicity
(ADCC), which is mediated by cytotoxic CD8+ T cells and natural
killer (NK) that carry membrane receptors for the Fc portion of
immunoglobulin G (IgG). Finally, activated macrophages carrying
Fc receptors may recognize and phagocyte erythrocytes opsonized
by autoantibodies and complement. While direct complementmediated lysis takes place mainly in the circulations and liver,
ADCC and phagocytosis occur preferentially in the spleen and
lymphoid organs [1].
AIHAs can be distinguished on the basis of the autoantibody Ig
class and thermal characteristics, i.e. the optimal temperature of reaction with autologous erythrocytes, in warm (wAIHA), cold (cold
agglutinin disease; CAD), and mixed forms, although atypical cases
of difficult diagnostic classification are reported with increasing
frequency [13]. Moreover, AIHAs are classified in primary (idiopathic), in which hemolysis dominates the clinical picture in the
absence of any other coexisting disorder, and in secondary forms,
accompanying and complicating an underlying disease. The majority of warm forms are idiopathic or associated with other autoimmune diseases; acute cold forms are mainly secondary to infections, whereas chronic CADs are frequently associated to lymphoproliferative or neoplastic diseases [13]. All these factors account for the great clinical heterogeneity of AIHAs, from
compensated forms without anemia to fulminating disease. Table1
shows the main characteristics of AIHAs.
288
Barcellini
289
Fig. 1. T-lymphocyte subsets and cytokine interactions in AIHA. Cytokines that were found elevated are highlighted in yellow: IL-2 and IL-12,
that induce the differentiation of CD4+ nave
T cells into Th1 subset, and IL-4, that promotes
Th2 switch. Elevated levels of TGF- favor the
differentiation of Th17 subset, which amplifies the
pro-inflammatory and autoimmune response. On
the contrary, decreased levels of both IFN- and
Tregs were found (highlighted in green). The
former resulted in decreased inhibition of Th2
response, i.e. an amplification of the autoantibodymediated autoimmune disease, and the latter may
cause a lack of down-regulation of inflammatory
and autoimmune pathways.
290
It is known that Tregs produce IL-10 and transforming growth factor (TGF-), and the IL-10/IL-12 imbalance may thus play an essential role in the onset and/or maintenance of AIHA. Several
other abnormalities of immunoregulatory cytokines have been reported in AIHA: IL-1, IL-2/IL-2R, IL-6, and IL-21 serum levels
were found increased; in culture experiments, the Th1 cytokines
IL-2 and IL-12 were reported elevated, whereas IFN- was found
reduced. Moreover, the Th2 cytokines IL-4 and IL-13 were reported increased, together with elevated production of IL-6, IL-10,
and IL-17 [36, 40, 41]. Patients with active hemolysis showed further reduction of IFN- and increased secretion of TGF-. Moreover, addition of IL-4, IL-6, IL-10, IL-13, and TGF- increased autoantibody secretion in culture [40]. All these data are in favor of a
reduced Th1- and a predominant Th2-like profile in the immunopathogenesis of AIHA, in contrast to the prevalent role of CD8+
IFN--secreting cytotoxic T cells in organ-specific autoimmunity
(i.e multiple sclerosis, experimental autoimmune encephalomyelitis, type 1 diabetes, and autoimmune thyroiditis). Moreover, elevated levels of TGF- favor the differentiation of a Th17 subset,
which amplifies the pro-inflammatory and autoimmune response.
On the contrary, decreased levels of both IFN- and Tregs resulted
in decreased inhibition of Th2 response, i.e. an amplification of the
autoantibody-mediated autoimmune disease (fig. 1). Notwithstanding these cytokine abnormalities, no difference was reported
in the phenotypic distributions of polymorphisms of the TNF-,
IL-10, IL-12, and CTLA-4 between AIHA patients and controls. As
regards therapy, immunoregulatory cytokines were evaluated after
low-dose rituximab (100 mg fixed dose for 4 weekly infusions)
[42]. The clinical response was correlated with an increase of IFN, and a reduction of IL-4 and IL-17, suggesting that the drug ex-
Barcellini
Pathogenic mechanism
Autoantibodies
IgG (all subclasses)
IgG1 and IgG3
IgM
Complement system
Cellular immunity
Immunoregulatory cytokines
291
in about one-third of cases, mainly warm IgG+C, mixed and atypical forms, with frequently associated reticulocytopenia [10]. As regards therapy, the majority of wAIHA patients received first-line
steroid therapy only, whereas patients with mixed and atypical
forms had an increased risk of relapse and were more frequently
treated with 2 or more therapy lines. Thrombotic events were associated with a severe onset, intravascular hemolysis and previous
splenectomy, and predictors of a fatal outcome were severe infections, particularly in splenectomized cases, acute renal failure,
Evans syndrome, and multi-treatment (4 or more lines) [10].
Moreover, we recently described 13 acute and very severe cases
of AIHA, displaying multi-drug refractoriness and related complications, of whom 7 were fatal. These challenging AIHAs showed a
highly unpredictable clinical courses, suggesting that rapidly evolving cases deserve a scrupulous attention by experienced clinicians
to avoid a delay in potentially life-saving strategies. In particular,
the presence of intravascular hemolysis, hemoglobinuria, DAT
positivity for IgG+C, reticulocytopenia, and/or Evans syndrome
should alarm clinicians to generously give steroid boluses, to early
utilize rituximab, and to consider plasma exchange in critical conditions. We also suggest performing splenectomy soon after rituximab failure, because of the poor prognosis of surgery after multiple
immunosuppressive therapies. Moreover, we suggest the use of
erythropoietin to reduce overtransfusion and an anti-thrombotic
prophylaxis, particularly in splenectomized patients [11]. Currently we use low-molecular-weight heparins at prophylactic doses,
i.e. 3050% of the anticoagulant dose, as long as the hemolytic process is active [51].
An additional factor that influence the clinical picture of AIHA
is the association with lymphoproliferative diseases. It is known
that about 10% of patients with chronic lymphocytic leukemia and
23% of those with non-Hodgkins and Hodgkins lymphoma develop AIHA. Moreover, in a population-based study of 86 patients
with CAD, a clonal bone marrow lymphoproliferation was demonstrated in 75% and 90% of the patients by bone marrow biopsy and
flow cytometry, respectively [20]. Moreover, there is increasing evidence that severe and fatal AIHA may be a complication of hematopoietic stem cell transplantation. A recent large study at Kings
College Hospital [52] described an overall incidence of 3.6% and
an association with hematopoietic stem cell transplantation from
unrelated donors. About two-thirds of cases required multiple
treatment, but only half achieved complete resolution of AIHA.
Ten cases were fatal, and death was directly attributable to AIHA
in 4 cases, indicating that post-transplantation AIHA had a higher
overall mortality (HR 2.48) than primary AIHA.
Finally, it is worth reminding the passenger lymphocyte syndrome, which is due to the production of antibodies directed
against recipient RBCs by passively transferred lymphocytes transfused with the stem cell, most often observed when the donor is
group O and the patient group A. A transient immune hemolysis
has also been associated with solid organ transplantation, with a
risk and degree of hemolysis in proportion to the mass of lymphocytes transplanted being lowest in kidney (antibody in 17%, hemolysis in 9%), next higher in liver (antibody in 40%, hemolysis in
29%), and highest in heart-lung transplants (antibody in 70%, hemolysis in 70%) [53, 54].
Conclusions
AIHA is an heterogeneous condition including warm, cold, and
mixed forms, along with atypical cases of difficult diagnostic classification (DAT-negative, warm IgM), which are reported with increasing frequency. It can be primary or secondary to lymphoproliferative syndromes, infections, immunodeficiency, and tumors.
Moreover, AIHA is described with increasing frequency following
hematopoietic stem cell transplantation, and in recipients of solid
organ transplants. The clinical presentation of primary AIHA may
vary from mild to very severe forms, with a reported mortality of
11% in older series, and of 4% in a more recent studies; secondary
forms are usually more severe, with an increased mortality, particularly in post-transplant forms. Several immunologic mechanisms are involved in the pathogenesis of AIHA: autoantibodies,
ADCC, phagocytes, B and T lymphocytes, Tregs, cytokines, and
the complement system. New insights in this complex scenario
may give hints for future therapeutic options specifically aimed at
correcting the main pathogenic mechanism involved in the different forms of AIHA.
Acknowledgement
This work was supported by research funding from Fondazione IRCCS Ca
Granda Ospedale Maggiore Policlinico, grant number RC 2014 and by Ministry
of Health, grant number RF 2010 convention N. 141/RF-20102303934.
Disclosure Statement
The author has no conflict of interest to declare.
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